No abstract

Fourier Transform Infrared Microspectroscopy (FTIRM) has been used to study the changes in mineral and matrix content and composition in replicate biopsies of nonosteoporotic human osteonal bone. Spectral maps in four orthogonal directions (in 10 microm steps) from the centers towards the peripheries of individual osteons were obtained from iliac crest biopsies of two necropsy cases. Mineral to matrix ratios, calculated from the ratio of integrated areas of the phosphate nu1,nu3 band at 900-1200 cm-1 to the amide I band at 1585-1725 cm-1, increased from the center to the periphery of the osteon. The total carbonate (based on the nu2 band at approximately 850-900 cm-1) to phosphate nu1,nu3 ratio decreased as the mineral to matrix ratio increased. Analysis of the nu2 CO32- band with a combination of second-derivative spectroscopy and curve fitting revealed a decrease in "labile" carbonate, a slight decrease in Type A and a slight increase in Type B carbonate from the center to the periphery of the osteon. Similar analysis of the components of the nu1,nu3 phosphate band with a combination of second-derivative spectroscopy and curve fitting revealed the presence of 11 major underlying moieties. These components were assigned by comparison with published frequencies for apatite and acid-phosphate containing calcium phosphates. The most consistent variations were alterations in the relative percent areas of bands at approximately 1020 and approximately 1030 cm-1, which had previously been assigned to nonstoichiometric and stoichiometric apatites, respectively. This ratio was used as an index of variation in crystal perfection throughout the osteon. This ratio decreased as the mineral to matrix ratio increased. The reproducibility of these parameters at multiple sites in multiple biopsies suggests their applicability for the analysis of mineral changes in disease.

Fourier transform infrared microspectroscopy (FTIRM) has been used to study the changes in mineral and matrix content and composition in replicate biopsies of nonosteoporotic human cortical and trabecular bone. Changes in osteonal bone in these same samples were reported previously. Spectral maps along and across the lamellae were obtained from iliac crest biopsies of two necropsy cases. Mineral:matrix ratios, calculated from the integrated areas of the phosphate nu1, nu3 band at 900-1200 cm-1 and the amide I band at approximately 1585-1725 cm-1, respectively, were relatively constant in both directions of analysis, i.e., along and across the lamellae. Analysis of the components of the nu1, nu3 phosphate band with a combination of second-derivative spectroscopy and curve fitting revealed the presence of 11 major underlying moieties. Of these, the ratio of the relative areas of the two underlying bands at approximately 1020 and approximately 1030 cm-1 has been shown to be a sensitive index of variation in crystal perfection in both human osteonal bone and in synthetic, poorly crystalline apatites. This ratio was calculated in both cortical and trabecular bone from human iliac crest biopsies along and across the lamellae. The ratio decreased, going from the periosteum to the medullary cavity in the cortical bone, and from the periphery towards the center of trabeculae. These observations were consistent within serial sections obtained from the same biopsy, multiple biopsies obtained from the same necropsy specimen, and biopsies obtained from the two different necropsy specimens. The results presented here along with previously reported changes in osteonal bone show a relation between bone age and "crystallinity/maturity" (a parameter dependent on crystallite size, hydroxyapatite-like stoichiometry, abundance of substituting ions such as CO32-; the more crystalline/mature, the more hydroxyapatite-like stoichiometry, the bigger the crystallite size, the less the ion substitution by ions such as CO32-) as deduced by the 1020/1030 cm-1 ratio. Invariably, younger normal bone is less mature/crystalline than older. These results provide a "baseline" for description of mineral properties, to which diseased bones may be compared.

Fourier Transform infrared spectroscopic analysis of maturing, poorly crystalline hydroxyapatite (HA) formed from the conversion of amorphous calcium phosphate (ACP) at constant pH or variable pH show only subtle changes in the v1, v3 phosphate absorption region (900 cm-1-1200 cm-1). This region is of interest because it can be detected by analysis of mineralized tissue sections using FT-IR microscopy. To evaluate the subtle spectral changes occurring during the maturation, second derivatives of the spectra were calculated. HA formed at constant pH showed little or no variation in the second derivative peak positions with bands occurring at 960 cm-1, 985 cm-1, 1030 cm-1, 1055 cm-1, 1075 cm-1, 1096 cm-1, 1116 cm-1, and 1145 cm-1. These bands can be assigned to molecular vibrations of the phosphate (PO4(3-)) moiety in an apatitic/stoichiometric environment of HA. In contrast, during the early stages of maturation of the HA formed at variable pH, second derivative peak positions occurring at 958 cm-1, 985 cm-1, 1020 cm-1, 1038 cm-1, 1112 cm-1, and 1127 cm-1 shifted in position with maturation, indicating that the environment of the phosphate species is changing as the crystals mature. Peaks at 1020 cm-1, 1038 cm-1, 1112 cm-1, and 1127 cm-1 were attributable to nonstoichiometry and/or the presence of acid phosphate-containing species. This concept was supported by the lower Ca:P molar ratios measured by chemical analysis of the synthetic material made at variable pH. Using the second derivative peak positions as initial input parameters, the v1, v3 phosphate region of the synthetic HAs prepared at constant pH were curve fit. X-ray diffraction patterns of these same materials were also curve fit to calculate the changes in crystallinty (size/perfection) in the c-axis 002 reflection as well as the 102, 210, 211, 112, 300, 202, and 301 planes. Linear regression analysis showed that the changes in the percent area of the underlying bands at 982 cm-1, 999 cm-1, 1030 cm-1, 1075 cm-1, 1096 cm-1, 1116 cm-1, and 1145 cm-1 were correlated with changes in crystallinity in one or more of the reflection planes. It is suggested that a combination of second-derivative and curve-fitting analysis of the v1, v3 phosphate contour allows the most reproducible evaluation of these spectra.

No abstract

Historically, osteoporosis has been defined as a disease in which there is "too little bone, but what there is, is normal." As a result of research design and sample selection limitations, published data contradict and confirm the historical definition. Because of these limitations, it has been hard to assess the contribution of mineral quality to mechanical properties, and to select therapeutic protocols that optimize bone mineral properties. The coupling of an optical microscope to an infrared spectrometer enables the acquisition of spectral data at known sites in a histologic section of mineralized tissue without loss of topography and/or orientation. The use of second-derivative spectroscopy coupled with curve-fitting techniques allows the qualitative and quantitative assessment of mineral quality (crystallite size and perfection, mineral:matrix ratio) at well-defined morphologic locations. We have previously applied these techniques to the study of normal human osteonal, cortical, and trabecular bone. The results indicated that the newly deposited bone mineral is less "crystalline/mature" than the older one. In the present study, Fourier transform infrared microspectroscopy (FTIRM) was applied to the study of human osteonal and cortical bone from iliac crest biopsies of untreated osteoporotic patients. The hypothesis tested was that osteoporotic bone mineral is monotonically different in its properties expressed as "crystallinity/maturity" than the normal. The results indicate significant differences in the mineral properties as expressed by crystal size and perfection, with the mineral from osteoporotic bone being more crystalline/mature than the normal.

No abstract

Fourier Transform Infrared Microspectroscopy (FTIRM) has been used to study the changes in mineral and matrix content and composition in replicate biopsies of nonosteoporotic human osteonal bone. Spectral maps in four orthogonal directions (in 10 microm steps) from the centers towards the peripheries of individual osteons were obtained from iliac crest biopsies of two necropsy cases. Mineral to matrix ratios, calculated from the ratio of integrated areas of the phosphate nu1,nu3 band at 900-1200 cm-1 to the amide I band at 1585-1725 cm-1, increased from the center to the periphery of the osteon. The total carbonate (based on the nu2 band at approximately 850-900 cm-1) to phosphate nu1,nu3 ratio decreased as the mineral to matrix ratio increased. Analysis of the nu2 CO32- band with a combination of second-derivative spectroscopy and curve fitting revealed a decrease in "labile" carbonate, a slight decrease in Type A and a slight increase in Type B carbonate from the center to the periphery of the osteon. Similar analysis of the components of the nu1,nu3 phosphate band with a combination of second-derivative spectroscopy and curve fitting revealed the presence of 11 major underlying moieties. These components were assigned by comparison with published frequencies for apatite and acid-phosphate containing calcium phosphates. The most consistent variations were alterations in the relative percent areas of bands at approximately 1020 and approximately 1030 cm-1, which had previously been assigned to nonstoichiometric and stoichiometric apatites, respectively. This ratio was used as an index of variation in crystal perfection throughout the osteon. This ratio decreased as the mineral to matrix ratio increased. The reproducibility of these parameters at multiple sites in multiple biopsies suggests their applicability for the analysis of mineral changes in disease.